Ribulose 1,5-bisphosphate (RuBP) carboxylase catalyzes the primary assimilation of carbon dioxide. Based on the stoichiometry of active-site modification with pyridoxal 5'-phosphate (PLP), we intend to investigate the regulation of the R. rubrum carboxylase. The PLP stoichiometry results suggest that the subunits of this enzyme communicate during catalysis. Thus, we will: (a) Determine the mechanism by which RuBP carboxylase interacts with substrates and effectors. (b) Determine if the CO2 activation site is distinct from the CO2 catalytic site. The fact that certain metabolites facilitate activation leads us to believe that we might be able to specifically covalently label both the catalytic and activation sites. Upon successful modification, peptides containing active site and activation site residues will be examined to gain direct chemical evidence for the similarity or distinctness of the sites. (c) Obtain direct chemical evidence for the similarity or distinctness of the catalytic subunits of Form I and Form II RuBP carboxylase from R. sphaeroides. (d) Begin to isolate intact chloroplasts to facilitate our projected carboxylase assembly studies.